The present invention relates to cationic polymer-nucleic acid compounds which have use in the delivery of nucleic acid to cells in biological systems, for instance in in vitro cell transfection research as well as in vivo delivery. The invention also relates to methods of mixing such compounds and potentially to gene therapy using such compounds.
The control of living processes is mediated through nucleic acids. Nucleic acids encode proteins which, as enzymes, hormones and other regulatory factors, carry out the processes which enable living organisms to function. Nucleic acids also encode for regulatory sequences which control the expression of proteins.
Because of its central role in living organisms, nucleic acids make an ideal therapeutic target. It is thought that many diseases could be controlled by the manipulation of nucleic acids in living organisms.
The key factor limiting therapies based on nucleic acid manipulation is the ability to deliver nucleic acids to the cells. Nucleic acids are fragile molecules which are highly negatively charged (one negative charge per phosphate group) and which are readily cleaved by nucleases present both in extracellular fluids and intracellular compartments. As a highly charged molecule it will not cross the lipid membranes surrounding the cell, nor can it readily escape from endosomal compartments involved in the uptake of macromolecules into cells. Even RNAi molecules, although smaller in molecular weight, show significant problems of stability and uptake.
Cationic lipid formulations suffer from a number of shortcomings. Cationic lipid formulations are unstable and have a relatively short shelf life. The short shelf life is at least partly due to the tendency of these formulations to aggregate.
The discovery of altering gene expressions by delivery to mammalian cells of nucleic acids such as plasmid DNA, double stranded DNA and small interference RNA (siRNA) offers the potential ability to treat a variety of diseases. However, therapeutic applications of this technology have been slow to materialize. A first obstacle is the targeting of a particular organ or tissue. It was thought that attaching specific ligands which have affinity to receptors of targeted cells to nucleic acids would solve the problem. However the number of suitable ligand/receptor combinations is limited, e.g. application of galactose-derived ligands to target hepatocytes. Additionally, receptor-mediated nucleic acid entry into cells is inevitably associated with endosomal formation and nucleic acid degradation in lysosomes. To prevent lysosome formation and nucleic acid degradation endosomolytic agents are commonly used. However, attaching molecules that facilitate endosome disruption and nucleic acid release (e.g. melittin-like peptide) cause severe toxicity.
Another obstacle is the high levels of nucleases in all extracellular compartments, causing degradation of nucleic acids injected subcutaneously or intravenously. Attaching nuclease-protective groups to nucleic acid complexes makes targeting and cell entry more difficult, and significantly increases the cost of the technology.
Complexation of nucleic acids with cationic liposomes offers some protection from nucleases and can facilitate fusion of a small portion of liposomes with cells which avoids lysosome formation. However, liposomal nucleic acid delivery lacks organ-specific targeting and mostly targets macrophages, yet results in toxicological effects due to the cationic surface charge.
As it stands today, there is no simple technology allowing nucleic delivery to cells in vivo. All attempts to deliver nucleic acids, more specifically, siRNA to cancer cells in vivo to knock down inhibitory checkpoint proteins failed, as did all attempts to deliver siRNA though blood-brain barrier to brain neurons.
We have developed new simple technology that overcomes the stated obstacles and sufficiently delivers nucleic acids to cells in normal organs as well as to cancer cells in experimental tumor models—in vivo.
It is an object of the invention to overcome at least some of the above problems.